The UDP-glucuronosyltransferases (UGT) are a family of enzymes involved in the glucuronidation of both endogenous agents such as bilirubin and steroids, as well as thousands of exogenous agents that are consumed in our diet or are administered as drugs. The UGTs are an important group of proteins, involved almost exclusively in the detoxification and eventual elimination of drugs and other compounds. Understanding the functional properties of these proteins, how their genes are regulated, as well as the genetic composition of the genes, is crucial to advancing our knowledge on the metabolism and excretion of drugs and xenobiotics. In rabbit, UGT cDNAs encoding the 4-nitrophenol UGTI*O6 and 4- hydroxybiphenyl (4-HBP) UGT2B13 have already been characterized in this laboratory. In addition, a cDNA encoding UGT2B14 and a cDNA that encodes a UGT in the UGT2C1 subfamily have also been characterized. As a result of the novel structural features of the UGTs, experiments are planned to construct highly enriched UGT cDNA libraries, which will be used to clone a series of additional UGTs. Based upon N-terminal analysis of purified proteins and similarities to known UGT amino sequence, results are presented in the Preliminary Results section of this application that argue for the feasibility of identifying the rabbit liver estrone and morphine UGT cDNAs. In addition, by using portions of the 5'-divergent regions of the UGT1, UGT2B and UGT2C cDNAs as probes, it is anticipated that additional novel UGT cDNA clones will be identified from the enriched UGT cDNA libraries. In order to investigate the potential substrate specificities of proteins encoded by these cDNAs, eukaryotic expression systems are being developed. For example, while the efficient expression of rabbit UGT1*06 and UGT2B13 can be demonstrated in transfected COS cells, preliminary results demonstrate the use baculovirus vectors and expression of UGT2B13 in Sf9 cells to be more efficient by an order of magnitude. To appreciate the catalytic properties of newly identified UGTs, the cDNAs will be placed under the control of baculovirus expression. With the ability to generate adequate amounts of protein, attempts will be made to document the types of substrates that interact with the different rabbit UDPGTs. One of the principal goals of this application is to examine the regulation and function of the different UGTs. Taking advantage of the unique physical properties of the different UGTs, with most transferases being divergent in the 5' region and highly conserved in the 3' region, selective antibodies to the UGTs will be developed using expression protein from the 5' regions of the cDNAs. It is anticipated that as our inventory of biologically useful reagents, such as cDNAs and antibodies, are characterized, it will be a natural outgrowth of these studies to utilize these reagents in experiments to characterize the properties of gene expression and protein expression in vivo.